Reaction mass of Chromate(1-), [N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthalenyl]acetamidato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-, hydrogen, compd. with N-cyclohexylcyclohexanamine (1:1) and hydrogen bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with dicyclohexylamine (1:1) and hydrogen bis[N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthyl]acetamidato(2-)]chromate(1-) , compound with dicyclohexylamine (1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay (Ames test, OECD TG 471): positive with and without metabolic activation

In vitro mutation study in mammalian cells (HPRT assay, OECD TG 476): negative with and without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Oct 2016 to 20 Oct 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test substance (as reported in the study report): Orasol Black X45
- Test substance No.: 16/0051-1
- Batch identification: 002-150504
- Content: 97.5 g/100 g (100 g/100 g minus water content)
- Homogeneity: Ensured by mixing before preparation of the test substance solutions.
- Storage stability: Guaranteed until 29 Apr 2025 as indicated by the sponsor

ADDITIONAL TEST SUBSTANCE INFORMATION:
- Date of production: 29 Apr 2015
- Physical state, appearance: solid, black
- Storage conditions: room temperature

Target gene:

his- (S. typhimurium), trp- (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and ß-naphthoflavone induced rat liver S-9 mix

Test concentrations with justification for top dose:

Doses for experiment 1: 0; 33; 100; 333; 1000; 2600 and 5200 μg/plate
Doses for experiment 2: 0; 1; 3.3; 10; 33; 100 and 333 μg/plate

Vehicle / solvent:

- Vehicle: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water. DMSO is suitable in bacterial reverse mutation tests also observed in historical control data which are available.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (2.5 µg/plate) + S9: TA1535, TA100, TA1537, TA98; 2-aminoanthracene (60 µg/plate) + S9: E. Coli; 4-nitro-o-phenylenediamine (10 µg/plate) - S9: TA98; N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (5 µg/plate) - S9: TA1535, TA100

Details on test system and experimental conditions:

STANDARD PLATE TEST
Test tubes containing 2-mL portions of soft agar were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
- 0.1 mL test solution or vehicle (negative control)
- 0.1 mL fresh bacterial culture
- 0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates within approx. 30 seconds.

After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants for S. typhimurium / trp+ revertants for E. Coli) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK).
Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

Evaluation criteria:

ACCEPTANCE CRITERIA:
Generally, the experiment was considered valid if the following criteria were met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- Fresh bacterial culture containing approximately 10E+9 cells per mL were used.

ASSESSMENT CRITERIA:
The test substance was considered positive in this assay if the following criteria were met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolising system.
A test substance was generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 100, TA 1537 and TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test up to the highest concentration.
- Strong test substance precipitation was found from about 333 μg/plate onward with and without S9 mix. Due to the precipitation an evaluation of the highest concentration (5200 μg/plate) was not possible in the 1st experiment.

Remarks on result:

other: 1st experiment at 1000 µg/plate

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to the read-across justification attached to section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With S9-mix: at the highest concentration 600 µg/mL Without S9-mix: at the highest concentration of 60 µg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Result read-across source CAS No. 12237-23-9

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo micronucleus test (OECD TG 474): no evidence for clastogenic or aneugenic effects.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to the read-across justification attached to section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

The high dose applied was toxic as manifested in the tolerability test by various symptoms

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Result read-across source CAS No. 12237-23-9

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

An ames test is available for the substance itself. As no mammalian mutation test is available for the substance itself a read-across is made to the HPRT test performed with a structural analogue of the substance.

Ames test (substance itself, EC 916-865-0)

The test item was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA), in a reverse mutation assay. The test item was tested using standard plate test (SPT) with and without metabolic activation (liver S9 mix from induced rats) at a dose range 1.0 μg - 5200 μg/plate. A relevant increase in the number of his+ or trp+ revertants (factor ≥2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥3: TA 1535 and TA 1537) was not observed in the standard plate test with or without metabolic activation using tester strain E. coli WP2 uvrA and with S9 mix using tester strain TA 1535. A distinct, reproducible and dose dependent increase in the number of his+ revertants exceeding a factor of 2 compared to the concurrent vehicle control was observed with TA 100 and TA 98 with and without metabolic activation. Under the experimental conditions of this study, the test item is strongly mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation (BASF, 2017).

 

HPRT assay (Orasol Black CN, CAS 12237-23-9)

The test item was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The test substance was suspended in DMSO. The cells were treated in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours. The results of each experiment were confirmed in a second and independent experiment (confirmatory experiment). The original experiment with metabolic activation was performed at the following concentrations: 22.22, 66.67, 200.0 and 600.0 µg/mL. The mean growth inhibiting values found at the highest concentration after treatment and expression were 63.88% and 18.35% respectively. In the confirmatory experiment the concentrations applied were 75.0, 150.0, 300.0 and 600.0 µg/mL. The highest concentration revealed a mean acute growth inhibition of 36.66%. The mean growth inhibitory effect after the expression period was 12.52%. N-Nitrosodimethylamine (DMN, 1.0 µ1/mL) was used as positive control. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test item revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG). The original experiment without metabolic activation was performed at the following concentrations: 2.22, 6.67, 20.0 and 60.0 µg/mL. The mean growth inhibition value found at the highest concentration after treatment was 99.83%. At subculture this concentration proved toxic. In the confirmatory experiment the concentrations applied were 6.25, 12.5, 25.0 and 50.0 µg/mL. The highest concentration revealed a mean acute growth inhibitory effect of 88.76%. The mean growth inhibition after the expression period was 18.73%. Ethylmethansulfonate (EMS, 0.3 µl/mL) was used as positive control. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-TG. Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that the test item and its metabolites did not show any mutagenic activity in this forward mutation system (Ciba Geiga Ltd, 1995).

Genetic toxicity in vivo

As no studies are available with the test substance itself a read-across is made to a structural analogue of the substance.

Mouse micronucleus test (Orasol Black CN, CAS 12237-23-9)

The test item, identified as a black-bluish powder, was investigated for clastogenic (and/or aneugenic) effects on mouse bone marrow cells in vivo. The test item was administered once by gavage to groups of 5 male and 5 female Tif MAGf (SPF) mice at doses of 800, 400 and 200 mg/kg bw. Additional groups of animals were treated with the vehicle alone or with the positive control cyclophosphamide (64 mg/kg bw). From the high dose group and from the negative control group animals were sacrificed 24 and 48 hours thereafter. From the intermediate and the low dose group and from the positive control group animals were sacrificed 24 hours after application. Subsequently femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for micronuclei. The high dose applied was toxic as manifested in the tolerability test by various symptoms. Higher doses could not be administered due to severe toxicity. In all dosage groups assessed at the different periods post treatment, no statistically significant increase in the number of micronucleated polychromatic erythrocytes was observed when compared with the respective negative control group. It is concluded that under the given experimental conditions no evidence for clastogenic or aneugenic effects was obtained in mice treated with the test item (Ciba Geiga Ltd, 1995).

Justification for classification or non-classification

Based on the available data, classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.